Nitric oxide (NO) is produced during acute cardiac allograft rejection by the expression of inducible nitric oxide synthase (iNOS). This three-year proposal will examine the significance of this pathophysiological production of NO. The primary hypothesis is that NO, alone or in combination with oxygen free-radicals, is a principal effector mechanism for: 1) the myocyte functional impairment seen in early rejection, and 2) the myocyte necrosis that develops as the rejection process progresses unabated. The corollary to this hypothesis is that specific iNOS inhibitors, alone or in combination with new oxygen free-radical scavengers, are potentially novel and non-immunosuppressive therapeutic options for acute allograft rejection. Two processes that occur during cardiac allograft rejection will be examined to test this hypothesis: 1) the necrosis of myocytes that occurs relatively late in rejection, and 2) the reversible contractile dysfunction that occurs earlier in rejection. The site(s) of INOS expression, and role of NO in cell necrosis and apoptosis will be studied. The role of superoxide and peroxynitrite in conjunction with NO will be examined. Interaction of NO with the L-type calcium channel as a cause of reversible contractile/electrophysiologic dysfunction will be examined. Application of novel iNOS inhibitors to ameliorate these pathophysiologic sequelae during acute allograft rejection is fundamental to these investigations. The experiments outlined in this proposal will apply functional, electrophysiological, histological, biochemical, immunohistochemical and molecular biological techniques to 1) isolated myocyte and papillary muscle preparations, and 2) vascularized rat allograft models. The coupling of available electrophysiologic and EPR techniques with direct NO quantitation using bioelectroanalytical techniques is unique. It is anticipated that this proposal will generate considerable insight into the effector role of NO in the setting of early cardiac allograft rejection, and will demonstrate the efficacy of iNOS inhibitors as potent and novel therapeutic options in preventing the sequelae of acute rejection.